Sex differences in secondary attack rate of IDDM to siblings of probands through older ages. Pittsburgh Etiology of IDDM Study.
To determine the descriptive epidemiological patterns of the secondary attack rate of insulin-dependent diabetes mellitus (IDDM) among siblings of probands through older ages. A family history analysis was performed on 1774 IDDM probands who were diagnosed or seen within 1 yr of diagnosis at Children's Hospital of Pittsburgh from 1 January 1950 through 31 December 1981. The probands were discharged on insulin and were diagnosed at less than 17 yr of age. The time frame permitted the risk of IDDM for siblings of probands to be calculated over a broad spectrum of age. Risk estimates for the 3966 full natural siblings through 10, 20, and 30 yr of age were 1.6, 4.1, and 6.3%, respectively. Secondary attack rates were equivalent for male and female siblings through 15 yr of age (3%); however, the risk to males increased an additional 4% between 16 and 30 yr of age compared with 2.5% for females (P = 0.01). There was no evidence of an excess sex concordance among affected sibling pairs. Males have a greater secondary attack rate of IDDM at older ages than females. This may be due to an increased exposure to environmental agents among males or protective influences operating among females.


Vascular Endothelial Cell Injury Is an Important Factor in the Development of Encapsulating Peritoneal Sclerosis in Long-Term Peritoneal Dialysis Patients.
Encapsulating peritoneal sclerosis (EPS) is a rare but serious and life-threatening complication of peritoneal dialysis (PD). However, the precise pathogenesis remains unclear; in addition, predictors and early diagnostic biomarkers for EPS have not yet to be established. Eighty-three peritoneal membrane samples taken at catheter removal were examined to identify pathological characteristics of chronic peritoneal deterioration, which promotes EPS in patients undergoing long-term PD treatment with low occurrence of peritonitis. According to univariable logistic regression analysis of the pathological findings, thickness of the peritoneal membrane (P = 0.045), new membrane formation score (P = 0.006), ratio of luminal diameter to vessel diameter (L/V ratio, P<0.001), presence of CD31-negative vessels (P = 0.021), fibrin deposition (P<0.001), and collagen volume fraction (P = 0.018) were associated with EPS development. In analyses of samples with and without EPS matched for PD treatment period, non-diabetes, and PD solution, univariable analysis identified L/V ratio (per 0.1 increase: odds ratio (OR) 0.44, P = 0.003) and fibrin deposition (OR 6.35, P = 0.027) as the factors associated with EPS. L/V ratio was lower in patients with fibrin exudation than in patients without fibrin exudation. These findings suggest that damage to vascular endothelial cells, as represented by low L/V ratio, could be a predictive finding for the development of EPS, particularly in long-term PD patients unaffected by peritonitis.


Island biogeography theory explains the genetic diversity of a fragmented rock ptarmigan (Lagopus muta) population.
The island biogeography theory is one of the major theories in ecology, and its applicability to natural systems is well documented. The core model of the theory, the equilibrium model of island biogeography, predicts that species diversity on an island is positively related to the size of the island, but negatively related by the island's distance to the mainland. In recent years, ecologists have begun to apply this model when investigating genetic diversity, arguing that genetic and species diversity might be influenced by similar ecological processes. However, most studies have focused on oceanic islands, but knowledge on how the theory applies to islands located on the mainland (e.g., mountain islands, forest islands) is scarce. In this study, we examined how the size and degree of isolation of mountain islands would affect the genetic diversity of an alpine bird, the rock ptarmigan (Lagopus muta). Within our study area, we defined the largest contiguous mountain area as the mainland, while smaller mountains surrounding the mainland were defined as islands. We found that the observed heterozygosity (H o) was significantly higher, and the inbreeding coefficient (F is) significantly lower, on the mainland compared to islands. There was a positive significant relationship between the unbiased expected heterozygosity (H n.b.) and island size (log km2), but a negative significant relationship between H o and the cost distance to the mainland. Our results are consistent with the equilibrium model of island biogeography and show that the model is well suited for investigating genetic diversity among islands, but also on islands located on the mainland.


Radial head arthroplasty with a modular metal spacer to treat acute traumatic elbow instability.
The use of a metal radial head prosthesis to help stabilize an elbow with traumatic instability is appealing because internal fixation of multifragment, displaced fractures of the radial head is susceptible to either early or late failure. The newer modular prostheses are easier to size and implant, but their effectiveness has not been investigated, to our knowledge. Twenty-seven patients in whom a radial head replacement with a modular metal spacer prosthesis had been performed to treat traumatic elbow instability were evaluated with use of the Mayo Elbow Performance Index (MEPI), the American Shoulder and Elbow Surgeons Elbow Evaluation Instrument (ASES), and the Disabilities of the Arm, Shoulder and Hand questionnaire (DASH). Radiographs were evaluated for arthrosis, periprosthetic radiolucency, and heterotopic ossification. Seven patients underwent one or more subsequent operations to treat residual instability, heterotopic ossification and elbow contracture, ulnar neuropathy, or a misplaced screw. In two of these patients, the prosthesis was removed as part of an elbow contracture release or to treat infection. At an average of forty months postoperatively, elbow motion in the entire group of twenty-seven patients averaged 131 degrees of flexion with a 20 degrees flexion contracture, 73 degrees of pronation, and 57 degrees of supination. Stability was restored to all twenty-seven elbows, and twenty-two patients had a good or excellent result according to the MEPI. Seventeen patients had radiographic evidence of lucency around the neck of the prosthesis that was not associated with increased pain, thirteen patients had clinically inconsequential heterotopic ossification anterior to the radial neck, and nine patients had radiographic changes in the capitellum. An intentionally loosely placed modular metal radial head prosthesis can help to restore stability in conjunction with repair of other fractures and reattachment of the lateral collateral ligament to the epicondyle in the setting of traumatic elbow instability with a comminuted fracture of the radial head. While a prosthesis that is too large can cause problems, lucencies around the stem of the intentionally loose prosthesis and most changes in the capitellum do not appear to cause problems, at least in the short term.


[Ovulation induction for in vitro fertilisation at the Tygerberg Hospital].
The protocol for in vitro fertilisation (IVF) at Tygerberg Hospital is presented and the results are analysed. Indications for ovulation induction for IVF included the following: (a) irreversible tubal damage; (b) infertility due to immunological factors; (c) male factor-infertility; and (d) endometriosis. A combination of human menopausal gonadotrophin (HMG) and clomiphene citrate (Clomid; Mer-National) and human chorionic gonadotrophin was used. Clomid is given in dosages of 100 mg for 5 days depending on the cycle length. Three doses of HMG (150 IU) are given on alternate days, starting on the second day of clomiphene treatment. If the leading follicle has not reached a mean diameter of 14 mm the day after the last HMG dose, another dose is given. This dosage is continued until the leading follicle reaches a diameter of 14 mm. A total of 109 cycles in 100 patients was analysed. Ova were considered to be mature as soon as the dominant follicle had reached a sonographic average diameter of 18 mm, another two follicles of 16 mm average diameter being present. Serum luteinising hormone levels were determined 4-hourly from the stage when the leading follicles exceeded an average diameter of 14 mm. In this study the pregnancy rate was 21,3% per laparoscopy and 24,4% per embryo transfer. The oestradiol levels on the 5th day of treatment have a predictive value of the length of stimulation.


The ORF2 protein of hepatitis E virus binds the 5' region of viral RNA.
Hepatitis E virus (HEV) is a major human pathogen in much of the developing world. It is a plus-strand RNA virus with a 7.2-kb polyadenylated genome consisting of three open reading frames, ORF1, ORF2, and ORF3. Of these, ORF2 encodes the major capsid protein of the virus and ORF3 encodes a small protein of unknown function. Using the yeast three-hybrid system and traditional biochemical techniques, we have studied the RNA binding activities of ORF2 and ORF3, two proteins encoded in the 3' structural part of the genome. Since the genomic RNA from HEV has been postulated to contain secondary structures at the 5' and 3' ends, we used these two terminal regions, besides other regions within the genome, in this study. Experiments were designed to test for interactions between the genomic RNA fusion constructs with ORF2 and ORF3 hybrid proteins in a yeast cellular environment. We show here that the ORF2 protein contains RNA binding activity. The ORF2 protein specifically bound the 5' end of the HEV genome. Deletion analysis of this protein showed that its RNA binding activity was lost when deletions were made beyond the N-terminal 111 amino acids. Finer mapping of the interacting RNA revealed that a 76-nucleotide (nt) region at the 5' end of the HEV genome was responsible for binding the ORF2 protein. This 76-nt region included the 51-nt HEV sequence, conserved across alphaviruses. Our results support the requirement of this conserved sequence for interaction with ORF2 and also indicate an increase in the strength of the RNA-protein interaction when an additional 44 bases downstream of this 76-nt region were included. Secondary-structure predictions and the location of the ORF2 binding region within the HEV genome indicate that this interaction may play a role in viral encapsidation.


Copper overload and deficiency both adversely affect the central nervous system of Drosophila.
The human copper homeostasis disorders Menkes and Wilson disease both have severe neurological symptoms. Menkes is a copper deficiency disorder whereas Wilson disease patients suffer from copper toxicity, indicating that tight control of neuronal copper levels is essential for proper nervous system development and function. Here we examine the consequences of neuronal copper deficiency and excess in the Drosophila melanogaster nervous system, using targeted manipulation of the copper uptake genes Ctr1A and Ctr1B and efflux gene ATP7 in combination with altered dietary copper levels. We find that pan-neuronal over expression of Ctr1B and ATP7 both result in a reduction in viability. The effects of Ctr1B over expression are exacerbated by dietary copper supplementation and rescued by copper limitation indicating a copper toxicity phenotype. Dietary manipulation has the opposite effect on ATP7 over expression, indicating that this causes neuronal copper deficiency due to excessive copper efflux. Copper deficiency also causes a highly penetrant developmental defect in surviving adult flies which can be replicated by both copper excess and copper deficiency targeted specifically to a small subset of neuropeptidergic cells. We conclude that both copper overload and excess have detrimental effects on Drosophila neuronal function, reducing overall fly viability as well as impacting on a specific neuropeptide pathway.


Association of CTR and COLIA1 alleles with BMD values in peri- and postmenopausal women.
The variability of bone mass and bone strength is in part genetically determined. The pathophysiology of the disease is complex and its heritability is almost certainly polygenic. In a large group of women from north eastern Italy, homogeneous for calcium intake and other risk factors for osteoporosis, we investigated three different genetic polymorphic markers that have been associated with bone mineral density (BMD). The study includes 663 postmenopausal (aged 48-85 years) and 52 perimenopausal (aged 47-53 years) women. Lumbar spine and hip BMD were measured by dual energy X-ray absorptiometry (DXA). After DNA extraction, the restriction enzymes utilized were MscI for the SP1 site of the collagen type I regulatory region (COLIA1), AluI for the calcitonin receptor (CTR) gene, and BsmI for the Vitamin D receptor (VDR) gene. COLIA1 genotype was significantly associated with age-adjusted hip BMD, with the highest values in the SS group and the lowest in the ss group (p < 0.05). The COLIA1 effect was not visible until the sixth decade of life, but it increased thereafter with aging, becoming statistically significant also at the lumbar spine in subjects aged >70 years. CTR genotype was also significantly related to bone mass in the CC group, with the lowest age and weight-adjusted BMD values at the spine (p < 0.05). The CTR genotype effect was greater in the younger subset of women. This suggests that the CTR genotype might influence the process of acquiring peak bone mass rather than the process of bone loss along aging. No trend association was found between BMD values and VDR genotype. These findings suggest an association between the COLIA1 gene polymorphism more with the age-related rate of bone loss than with peak bone mass, which apparently is somewhat affected by CTR gene polymorphism.


Secreted frizzled-related protein 4 inhibits proliferation and metastatic potential in prostate cancer.
Secreted frizzled-related proteins (sFRP4) inhibits Wnt signaling and thus cellular proliferation in androgen-independent prostate cancer cells in vitro. However, increased expression of membranous sFRP4 is associated with a good prognosis in human localized androgen-dependent prostate cancer, suggesting a role for sFRP4 in early stage disease. Here, we investigated the phenotype of sFRP4 overexpression in an androgen-dependent prostate cancer model. An sFRP4-overexpressing androgen-dependent (LNCaP) prostate cancer model was established to assess changes in cellular proliferation, the expression, and subcellular localization of adhesion molecules and cellular invasiveness, and compared with the findings in sFRP4-overexpressing androgen-independent cells (PC3). sFRP4 overexpression in both cell line models resulted in a morphologic change to a more epithelioid cell type with increased localization of beta-catenin and cadherins (E-cadherin in LNCaP, N-cadherin in PC3) to the cell membrane. Functionally, sFRP4 overexpression was associated with a decreased rate of proliferation (P = 0.0005), decreased anchorage-independent growth (P < 0.001), and decreased invasiveness in PC3 cells (P < 0.0001). Furthermore, increased membranous sFRP4 expression was associated with increased membranous beta-catenin expression (P = 0.02) in a cohort of 224 localized human androgen-dependent prostate cancers. These data suggest that sFRP4 is an inhibitor of prostate cancer growth and invasion in vitro independent of androgen receptor (AR) signaling with correlative evidence in human androgen-dependent disease suggesting similar changes in the clinical setting. Consequently, potential therapeutic strategies to modulate Wnt signaling by sFRP4 will be relevant to both localized androgen-dependent prostate cancer and advanced metastatic disease.


Lifetime methamphetamine dependence is associated with cerebral microgliosis in HIV-1-infected adults.
Methamphetamine (Meth) use is common among HIV-infected persons. It remains unclear whether Meth dependence is associated with long-lasting degenerative changes in the brain parenchyma and microvasculature of HIV-infected individuals. We examined the postmortem brains of 78 HIV-infected adults, twenty of whom were diagnosed with lifetime Meth dependence (18 past and two current at the final follow-up visit). Using logistic regression models, we analyzed associations of Meth with cerebral gliosis (immunohistochemistry for ionized calcium-binding adapter molecule-1 (Iba1) and glial fibrillary acidic protein (GFAP) in frontal, temporo-parietal, and putamen-internal capsule regions), synaptodendritic loss (confocal microscopy for synaptophysin (SYP) and microtubule-associated protein-2 (MAP2) in frontal cortex), β-amyloid plaque deposition (immunohistochemistry in frontal and temporo-parietal cortex and putamen), and arteriolosclerosis (histopathology in forebrain white matter). We found that Meth was associated with marked Iba1 gliosis in the temporo-parietal region (odds ratio, 4.42 (95 % confidence interval, 1.36, 14.39), p = 0.014, n = 62), which remained statistically significant after adjusting for HIV encephalitis, white matter lesions, and opportunistic diseases (n = 61); hepatitis C virus seropositivity (n = 54); and lifetime dependence on alcohol, opiates, and cannabis (n = 62). There was no significant association of Meth with GFAP gliosis, SYP or MAP2 loss, β-amyloid plaque deposition, or arteriolosclerosis. In conclusion, we found lifetime Meth dependence to be associated with focal cerebral microgliosis among HIV-infected adults, but not with other brain degenerative changes examined. Some of the changes in select brain regions might be reversible following extended Meth abstinence or, alternatively, might have not been induced by Meth initially.


Systematic control of the rate of singlet fission within 6,13-diphenylpentacene aggregates with PbS quantum dot templates.
Lead chalcogenide quantum dots (QDs) are promising acceptors for photovoltaic devices that harness the singlet fission (SF) mechanism. The rate of singlet fission of polyacenes in the presence of QDs is a critical parameter in determining the performance of such devices. The present study demonstrates that the rates of SF in a pentacene derivative, 6,13-diphenylanthracene (DPP), are modulated by forming coaggregates with PbS QDs in aqueous dispersions. PbS QDs generally accelerate SF within DPP aggregates, and the extent of acceleration depends on the size of the QD. The average rate of SF increases from 0.074 ps-1 for DPP-only aggregates to 0.37 ps-1 within DPP-D co-aggregates for QDs with radius 2.2 nm, whereas co-aggregation with the smallest (r = 1.6 nm) and largest (r = 2.7 nm) QDs we tried only slightly change the SF rate. The rate variation is associated with (i) the density of surface ligands, which is influenced by the faceting of the PbS surface, and (ii) the local dielectric constant for DPP. To accelerate SF, the ligands should be dense enough to provide sufficient affinity for DPP aggregates and effectively perturb the perpendicular alignment of DPP monomers within aggregates to increase the intermolecular coupling that promotes SF, but should not be too dense so as to form a low dielectric environment that disfavors SF. The study suggests that it is critical to consider the influence of the microenvironment of the QD surface on photophysical processes when fabricating QD/organic hybrid devices.


Risks from disease caused by Mycobacterium orygis as a consequence of Greater one-horned Rhinoceros (Rhinoceros unicornis) translocation in Nepal.
The greater one-horned rhinoceros (Rhinoceros unicornis) is listed as vulnerable by the IUCN Red List. Mycobacterium orygis-associated disease was identified in a single greater one-horned rhino in Chitwan National Park in February 2015 prior to a planned translocation of five greater one-horned rhinoceros from Chitwan National Park to Bardia National Park for conservation purposes. This paper describes a qualitative disease risk analysis conducted retrospectively post-translocation for Mycobacterium orygis and this translocation, with the aim to improve the understanding of disease threats to the conservation of greater one-horned rhino. The disease risk analysis method used was devised by Sainsbury & Vaughan-Higgins (Conservation Biology, 26, 2017, 442) with modifications by Bobadilla Suarez et al (EcoHealth, 14, 2017, 1) and Rideout et al (EcoHealth, 14, 2017, 42) and included the use of a scenario tree and an analysis of uncertainty as recommended by Murray et al. (Handbook on import risk analysis for animals and animal products. Volume 1. Introduction and qualitative risk analysis, 2004), and the first time this combination of methods has been used to assess the risk from disease in a conservation translocation. The scenario tree and analysis of uncertainty increased the clarity and transparency of the analysis. Rideout et al.'s (EcoHealth, 14, 2017, 42) criteria were used to assess the source hazard and may be useful in comparative assessment of source hazards for future conservation translocations. The likelihood of release into the destination site of Mycobacterium orygis as a source hazard was estimated as of low risk, the risk of exposure of populations at the destination was of high risk and the likelihood of biological and environmental consequences was low. Overall, the risk from disease associated with Mycobacterium orygis as a result of this translocation was found to be low. Recommendations on disease risk management strategies could be improved with a better understanding of the epidemiology including the presence/absence of Mycobacterium orygis in greater one-horned rhino to develop effective disease risk management strategies.


Shifts in the rumen microbiota due to the type of carbohydrate and level of protein ingested by dairy cattle are associated with changes in rumen fermentation.
Balancing energy and nitrogen in the rumen is a key to both profitability and environmental sustainability. Four dairy cows were used in a Latin square experimental design to investigate the effect of severe nitrogen underfeeding (110 vs. 80% of requirements) and the type of carbohydrate consumed [neutral detergent fiber rich (FIB) vs. starch rich (STA)] on the rumen ecosystem. These dietary treatments modified both rumen fermentation and microbial populations. Compared with STA diets, consumption of FIB diets increased bacterial and fungal diversity in the rumen and also increased the concentrations of cellulolytic microorganisms, including protozoa (+38%), anaerobic fungi (+59%), and methanogens (+27%). This microbial adaptation to fiber utilization led to similar digestibility values for the 2 carbohydrate sources and was accompanied by a shift in the rumen fermentation patterns; when the FIB diets were consumed, the cows had greater ruminal pH, ammonia concentrations, and molar proportions of acetate and propionate compared with when they consumed the STA diets. Certain rumen microorganisms were sensitive to a shortage of nitrogen; rumen concentrations of ammonia were 49% lower when the low-protein (LP) diets were consumed as were total bacteria (-13%), anaerobic fungi (-28%), methanogens (-27%), protozoa (-19%), cellulolytic bacteria, and microbial diversity compared with when the high-protein (HP) diets were consumed. As a result, the digestibility of the LP diets was less than that of the HP diets. These findings demonstrated that the rumen microbial ecosystem is directly linked to the rumen fermentation pattern and, to some extent, to the efficiency of diet utilization by dairy cattle.


Species, sex, size and male maturity composition of previously unreported elasmobranch landings in Kuwait, Qatar and Abu Dhabi Emirate.
This paper presents data from the first major survey of the diversity, biology and fisheries of elasmobranchs in the Persian (Arabian) Gulf. Substantial landings of elasmobranchs, usually as gillnet by-catch, were recorded in Kuwait, Qatar and the Emirate of Abu Dhabi (part of the United Arab Emirates), although larger elasmobranchs from targeted line fisheries were landed in Abu Dhabi. The elasmobranch fauna recorded was distinctive and included species that are undescribed, rare and have a highly restricted known distribution. Numerical abundance was dominated by sharks (c. 80%), of which carcharhinids were by far the most important. The milk shark Rhizoprionodon acutus and whitecheek shark Carcharhinus dussumieri together comprised just under half of all recorded individuals. Around 90% of recorded sharks were small (50-90 cm total length, L(T) ) individuals, most of which were mature individuals of species with a small maximum size (<100 cm L(T) ), although immature individuals of larger species (e.g. Carcharhinus sorrah and other Carcharhinus spp.) were also important. The most frequently recorded batoid taxa were cownose rays Rhinoptera spp., an undescribed whipray, and the granulated guitarfish Rhinobatos granulatus. The first size, sex and maturity data for a wide range of Gulf elasmobranch species are presented (including L(T) at 50% maturity for males of four shark species) and include some notable differences from other locations in the Indo-West Pacific Ocean. A number of concerns regarding the sustainability of the fishery were highlighted by this study, notably that most of the batoid species recorded are classed by the IUCN Red List as vulnerable, endangered, data deficient or not evaluated. Despite their considerable elasmobranch landings, none of the three countries sampled have developed a 'Shark Plan' as encouraged to do so under the FAO International Plan of Action: Sharks. Furthermore, Kuwait and Qatar currently report zero or no elasmobranch landings to the FAO.


Conserving Phylogenetic Diversity Can Be a Poor Strategy for Conserving Functional Diversity.
For decades, academic biologists have advocated for making conservation decisions in light of evolutionary history. Specifically, they suggest that policy makers should prioritize conserving phylogenetically diverse assemblages. The most prominent argument is that conserving phylogenetic diversity (PD) will also conserve diversity in traits and features (functional diversity [FD]), which may be valuable for a number of reasons. The claim that PD-maximized ("maxPD") sets of taxa will also have high FD is often taken at face value and in cases where researchers have actually tested it, they have done so by measuring the phylogenetic signal in ecologically important functional traits. The rationale is that if traits closely mirror phylogeny, then saving the maxPD set of taxa will tend to maximize FD and if traits do not have phylogenetic structure, then saving the maxPD set of taxa will be no better at capturing FD than criteria that ignore PD. Here, we suggest that measuring the phylogenetic signal in traits is uninformative for evaluating the effectiveness of using PD in conservation. We evolve traits under several different models and, for the first time, directly compare the FD of a set of taxa that maximize PD to the FD of a random set of the same size. Under many common models of trait evolution and tree shapes, conserving the maxPD set of taxa will conserve more FD than conserving a random set of the same size. However, this result cannot be generalized to other classes of models. We find that under biologically plausible scenarios, using PD to select species can actually lead to less FD compared with a random set. Critically, this can occur even when there is phylogenetic signal in the traits. Predicting exactly when we expect using PD to be a good strategy for conserving FD is challenging, as it depends on complex interactions between tree shape and the assumptions of the evolutionary model. Nonetheless, if our goal is to maintain trait diversity, the fact that conserving taxa based on PD will not reliably conserve at least as much FD as choosing randomly raises serious concerns about the general utility of PD in conservation.


Cutting edge: anti-CD154 therapeutic antibodies induce infectious transplantation tolerance.
Nondepleting anti-CD154 (CD40 ligand) mAbs have proven effective in inducing transplantation tolerance in rodents and primates. In the induction phase, anti-CD154 Ab therapy is known to enhance apoptosis of Ag reactive T cells. However, this may not be the sole explanation for tolerance, as we show in this study that tolerance is maintained through a dominant regulatory mechanism which, like tolerance induced with CD4 Abs, manifests as infectious tolerance. Therefore, tolerance induced with anti-CD154 Abs involves not only the deletion of potentially aggressive T cells, but also a contagious spread of tolerance to new cohorts of graft-reactive T cells as they arise.


Mortality from leukemia and other diseases among workers at a petroleum refinery.
This study evaluates the mortality experience of 9484 white men who worked at a petroleum refinery. We compared the numbers of deaths among these men during the period 1940 through 1984 with the numbers expected on the basis of the mortality rates of US white men. Overall, there were 2874 observed compared with 3568 expected deaths (standardized mortality ratio, 77). Mortality rates for most major cause of death categories and most cancers were also lower than expected. However, there was a statistically significant 50% excess of leukemia deaths (44 observed/29.6 expected). Lymphocytic leukemia was increased both among men hired before 1940 and among men hired in 1940 or later. In contrast, myelocytic leukemia was increased only among men hired in 1940 or later. The restriction of the myelocytic leukemia excess to men hired in 1940 or later may be related to process changes which occurred at the refinery after 1940 and which resulted in an increased percentage of benzene in certain refinery streams. The presence of an excess of lymphocytic leukemia, but not myelocytic leukemia, among men hired before 1940 suggests that some factor other than benzene was responsible for the former condition.


Population risk and burden of health behavioral-related all-cause, premature, and amenable deaths in Ontario, Canada: Canadian Community Health Survey-linked mortality files.
To examine the association of all-cause and premature mortality with four modifiable lifestyle behaviors and quantify the burden of behavioral-related premature death in Ontario, Canada. We analyzed a cohort of 149,262 adults in the 2000-2010 Canadian Community Health Surveys, linked to vital statistics data to ascertain deaths until December 31, 2015. The strength of the association between behaviors (smoking, body mass index, physical inactivity, and alcohol consumption) and all-cause and premature mortality was estimated using sex-specific Cox proportional hazards models. We estimated the proportion of deaths from causes amenable to the health system by behavior. After full adjustment, hazard ratios (95% confidence interval) for premature mortality were significantly increased for heavy smokers versus nonsmokers [males: 5.48 (4.55-6.60); females 4.45 (3.49-5.66)]; obese class III versus normal weight [males: 2.47 (1.76-3.48); females: 1.73 (1.29-2.31)]; and physically inactive versus active [males: 1.25 (1.07-1.45); females: 1.70 (1.41-2.04)]. In both sexes, a disproportionate burden of amenable deaths were experienced by heavy smokers, severely obese, physically inactive, and heavy drinkers. The findings emphasize the importance of prevention to reduce the prevalence of risk behaviors that contribute to a large burden of premature deaths that are amenable to the health system.


Surface Treatment on Physical Properties and Biocompatibility of Orthodontic Power Chains.
The conventional orthodontic power chain, often composed of polymer materials, has drawbacks such as a reduction of elasticity owing to water absorption as well as surface discoloration and staining resulting from food or beverages consumed by the patient. The goal of this study was to develop a surface treatment (nanoimprinting) for orthodontic power chains and to alleviate their shortcomings. A concave template (anodic alumina) was manufactured by anodization process using pure aluminum substrate by employing the nanoimprinting process. Convex nanopillars were fabricated on the surface of orthodontic power chains, resulting in surface treatment. Distinct parameters of the nanoimprinting process (e.g., imprinting temperature, imprinting pressure, imprinting time, and demolding temperature) were used to fabricate nanopillars on the surface of orthodontic power chains. The results of this study showed that the contact angle of the power chains became larger after surface treatment. In addition, the power chains changed from hydrophilic to hydrophobic. The power chain before surface treatment without water absorption had a water absorption rate of approximately 4%, whereas a modified chain had a water absorption rate of approximately 2%-4%. Furthermore, the color adhesion of the orthodontic power chains after surface modification was less than that before surface modification.


Cloning of two human homologs of the Drosophila single-minded gene SIM1 on chromosome 6q and SIM2 on 21q within the Down syndrome chromosomal region.
As part of our effort to clone genes of human chromosome 21 that may contribute to Down syndrome, we have previously isolated four exons with homology to Drosophila single-minded (sim) gene, which encodes a transcription factor that is a master regulator of fruit fly neurogenesis. These exons were used to clone and characterize two human homologs of the Drosophila sim gene, SIM1 and SIM2, which map to chromosomes 6q16.3-q21 and 21q22.2, respectively; SIM2 maps within the so-called Down syndrome chromosomal region. Recently, two mouse homologs, Sim1 and Sim2, also have been identified. There is a high level of homology among human, mouse, and Drosophila sim genes in their amino-terminal half where the conserved bHLH, PAS1, PAS2, and HST domains are present. In contrast, the carboxy-terminal parts are only homologous between SIM1 and Sim1 and SIM2 and Sim2. Two isoforms (SIM2 and SIM2s) of human SIM2 have been detected that differ in their 3' ends. Northern blot analysis revealed one mRNA SIM1 species of approximately 9.5 kb and four different mRNA SIM2 species of 2.7, 3, 4.4, and 6 kb in human fetal kidney. The function of both human SIM1 and SIM2 is unknown. However, three copies of SIM2 may contribute to some specific Down syndrome phenotypes because of (1) mapping position, (2) potential function as transcriptional repressor, (3) likely dimerization with other transcription factors, (4) the temporal and spatial expression pattern of mouse Sim2, and (5) the potentially analogous role of human SIM2 to that of Drosophila sim during neurogenesis.


Simplified low-cost production of O-antigen from Salmonella Typhimurium Generalized Modules for Membrane Antigens (GMMA).
The Novartis Vaccines Institute for Global Health is developing vaccines using outer membrane particles, known as Generalized Modules for Membrane Antigens (GMMA). These are blebs of outer membrane and periplasm, shed from the surface of living Gram-negative bacteria following the targeted deletion of proteins involved in maintaining the integrity of the inner and outer membranes. The current study investigates the use of GMMA as starting material for extraction of membrane components, focusing on the O-antigen polysaccharide portion of lipopolysaccharide from Salmonella Typhimurium. We show that the amount of O-antigen extracted from GMMA by acid hydrolysis is comparable to the quantity extracted from whole wild type bacteria, but with less protein and DNA contaminants. Compared to conventional purification, GMMA enabled a reduction in the number of purification steps required to obtain the O-antigen polysaccharide with the same purity. Purification processes from GMMA and bacteria were characterised by similar final yields. Use of GMMA as starting material provides the possibility to simplify the purification process of O-antigen, with a consequent decrease in manufacturing costs of O-antigen-based glyconjugate vaccines against Salmonella strains and potentially other Gram-negative bacteria.


Convergence of longevity and immunity: lessons from animal models.
An increasing amount of data implicate immunity-mostly innate immunity-in the ageing process; both during healthy ageing as well as in neurodegenerative diseases. Despite the aetiology however, the underlying mechanisms are poorly understood. Here we review what we know from model organisms (worms, flies and mice) on the possible mechanistic details that connect immunity and ageing. These links provide evidence that inter-tissue communication (especially the interaction between gut and brain), hormonal control mechanisms and intestinal microbiota determine immune system activity and thus influence lifespan.


A family practice model of health care for homeless people: collaboration with family nurse practitioners.
The growth of a large population of people without permanent housing in the United States has brought with it the necessity to address the unmet health needs of this group. As homelessness spreads, its demographic pattern has become more heterogeneous, with young vulnerable families now the major subgroup. This paper explores issues of homelessness in Westchester County, N.Y., which, despite being the 10th richest county in the United States, has the highest per capita homeless rate in the state. Children younger than age 18 represent the majority of this group. Barriers to the delivery of health care services are described, including fragmented life-styles, lack of insurance, insensitivity of care givers, distance from services, and inflexibility of traditional sources of health care. A model that has been developed for delivery of services is discussed. This model, the Outreach Health Care Unit, is run by nurse practitioners in collaboration with family physicians and is centered at the site of social service activities for homeless families and single men in Yonkers, N.Y. It is a collaborative endeavor of a nursing school, community hospital family practice residency program, and a network of social service agencies. The use of this model for education and research is also discussed since the goal is to provide both health services and training for health care providers.


ADSSL1 mutation relevant to autosomal recessive adolescent onset distal myopathy.
Distal myopathy is a heterogeneous group of muscle diseases characterized by predominant distal muscle weakness. A study was done to identify the underlying cause of autosomal recessive adolescent onset distal myopathy. Four patients from 2 unrelated Korean families were evaluated. To isolate the genetic cause, exome sequencing was performed. In vitro and in vivo assays using myoblast cells and zebrafish models were performed to examine the ADSSL1 mutation causing myopathy pathogenesis. Patients had an adolescent onset distal myopathy phenotype that included distal dominant weakness, facial muscle weakness, rimmed vacuoles, and mild elevation of serum creatine kinase. Exome sequencing identified completely cosegregating compound heterozygous mutations (p.D304N and p.I350fs) in ADSSL1, which encodes a muscle-specific adenylosuccinate synthase in both families. None of the controls had both mutations, and the mutation sites were located in well-conserved regions. Both the D304N and I350fs mutations in ADSSL1 led to decreased enzymatic activity. The knockdown of the Adssl1 gene significantly inhibited the proliferation of mouse myoblast cells, and the addition of human wild-type ADSSL1 reversed the reduced viability. In an adssl1 knockdown zebrafish model, muscle fibers were severely disrupted, which was evaluated by myosin expression and birefringence. In these conditions, supplementing wild-type ADSSL1 protein reversed the muscle defect. We suggest that mutations in ADSSL1 are the novel genetic cause of the autosomal recessive adolescent onset distal myopathy. This study broadens the genetic and clinical spectrum of distal myopathy and will be useful for exact molecular diagnostics.


Atorvastatin does not exhibit antiviral activity against HCV at conventional doses: a pilot clinical trial.
Cholesterol biosynthesis is an integral part of HCV RNA replication. Not only does HCV RNA replicate on lipid rafts, but it also requires cholesterol intermediates to replicate. In addition, it has been shown in vitro that several HMG-CoA reductase inhibitors can decrease HCV RNA replication by > or = 1 log. Therefore, we designed a clinical trial to evaluate the effect of atorvastatin on HCV RNA levels. In this prospective clinical trial, where patients served as their own control, 10 HCV-infected patients who required treatment for high cholesterol were given 20 mg atorvastatin per day. Although serum cholesterol and LDL predictably decreased significantly, there was no statistically significant change in week 4 and week 12 HCV RNA levels compared to pretreatment HCV RNA levels by the paired Student t test. It is unclear whether the addition of an HMG-CoA reductase inhibitor to interferon or a more potent inhibitor of cholesterol biosynthesis may be required to inhibit HCV RNA replication in vivo. In conclusion, atorvastatin, and likely all HMG-CoA reductase inhibitors, does not inhibit HCV RNA replication in vivo at conventional doses.


A case of coexistent angiosarcoma and follicular carcinoma of the thyroid.
Angiosarcoma of the thyroid has long been a controversial entity, and it is histologically defined as cleft-like anastosmosing spaces lined by large, atypical cells of endothelial lineage. However, clear-cut separation between the angiosarcoma and anaplastic carcinoma of the thyroid is difficult because they yield nearly the same clinical prognosis and overlapping histologic findings. We report a case of thyroid neoplasm composed of minimally invasive well differentiated follicular carcinoma and angiosarcoma with intervening transitional area. Immunohistochemically, the angiosarcomatous portion showed focal immunoreactivity for endothelial markers such as CD31, CD34, Ulex europaeus 1 lectin, factor VIII-related antigen, and immunonegativity for epithelial markers including pancytokeratin, epithelial membrane antigen and thyroglobulin, whereas the reverse was demonstrated in the minimally invasive follicular carcinomatous portion. The follicular carcinoma portion was positive for thyroid transcription factor-1 (TTF-1). Each component showed ultrastructural findings of epithelial and endothelial differentiation, respectively. The present case was unique in that angiosarcoma of the thyroid was confirmed by immunohistochemistry and electron microscopy, as well as light microscopy, and also coexisted with a minimally invasive well differentiated follicular carcinoma in the same mass. This combination has never been described in the literature. Although restricted to a single case, the present case further supports that angiosarcoma is a true existent entity rather than a variant of anaplastic carcinoma.


Efficacy and safety of efavirenz 400 mg daily versus 600 mg daily: 96-week data from the randomised, double-blind, placebo-controlled, non-inferiority ENCORE1 study.
The week 48 primary analysis of the ENCORE1 trial established the virological non-inferiority and safety of efavirenz 400 mg compared with the standard 600 mg dose, combined with tenofovir and emtricitabine, as first-line HIV therapy. This 96-week follow-up of the trial assesses the durability of efficacy and safety of this treatment over 96 weeks. ENCORE1 was a double-blind, placebo-controlled, non-inferiority trial done at 38 clinical sites in 13 countries. HIV-infected adult patients (≥16 years of age) with no previous antiretroviral therapy, a CD4 cell count of 50-500 cells per μL, and plasma HIV-1 viral load of at least 1000 copies per mL were randomly assigned (1:1) by an electronic case report form to receive fixed-dose daily tenofovir 300 mg and emtricitabine 200 mg plus efavirenz either 400 mg daily or 600 mg daily. Participants, physicians, and all other trial staff were masked to treatment assignment. Randomisation was stratified by HIV-1 viral load at baseline (≤ or >100 000 copies per mL). The primary endpoint was the difference in the proportions of patients in the two treatment groups with a plasma HIV-1 viral load below 200 copies per mL at week 96. Treatment groups were deemed to be non-inferior if the lower limit of the 95% CI for the difference in viral load was above -10% by modified intention-to-treat analysis. Non-inferiority was assessed in the modified intention-to-treat, per-protocol, and non-completer=failure (NC=F) populations. Adverse events and serious adverse events were summarised by treatment group. This study is registered with ClinicalTrials.gov, number NCT01011413. Between Aug 24, 2011, and March 19, 2012, 636 eligible participants were enrolled and randomly assigned to the two treatment groups (324 to efavirenz 400 mg and 312 to efavirenz 600 mg). The intention-to-treat population who received at least one dose of study drug comprised 630 patients: 321 in the efavirenz 400 mg group and 309 in the efavirenz 600 mg group. 585 patients (93%; 299 in the efavirenz 400 mg group and 286 in the 600 mg group) completed 96 weeks of follow-up. At 96 weeks, 289 (90·0%) of 321 patients in the efavirenz 400 mg group and 280 (90·6%) of 309 in the efavirenz 600 mg group had a plasma HIV-1 viral load less than 200 copies per mL (difference -0·6, 95% CI -5·2 to 4·0; p=0·72), which suggests continued non-inferiority of the lower efavirenz dose. Non-inferiority was recorded for thresholds of less than 50 and less than 400 copies per mL, irrespective of baseline plasma viral load. Adverse events were reported by 291 (91%) of 321 patients in the efavirenz 400 mg group and by 285 (92%) of 309 in the 600 mg group (p=0·48). The proportions of patients reporting an adverse event that was definitely or probably related to efavirenz were 126 (39%) for efavirenz 400 mg and 148 (48%) for efavirenz 600 mg (p=0·03). The number of patients who reported serious adverse events did not differ between the groups (p=0·20). Our findings confirm that efavirenz 400 mg is non-inferior to the standard dose of 600 mg in combination with tenofovir and emtricitabine as initial HIV therapy over 96 weeks. Fewer efavirenz-related adverse events were reported with the 400 mg efavirenz dose than with the 600 mg dose. These findings support the routine use of efavirenz 400 mg. The coadministration of rifampicin and efavirenz 400 mg needs further investigation. Bill & Melinda Gates Foundation, and UNSW Australia.


The Humber catchment and its coastal area: from UK to European perspectives.
The present water quality of the Humber rivers and coastal zone depends on a complex interplay of factors, including physical ones, such as the underlying geology, which influences soil type, climatic ones, such as the rainfall, which influences runoff, socio-economic ones, which influence present-day human activities in the catchment, and the legacy of former activities, such as contaminated sediments from mining. All of these factors affect the fluxes of nutrients and other contaminants to the rivers and coastal zone. The Water Framework Directive (WFD) requires the production of a river basin management plan intended to lead to the achievement of good chemical and ecological status for all water bodies in the catchment over the next two decades. This paper provides an overview of the current environmental and socio-economic state of the Humber catchment and coastal zone, and broadly examines how socio-economic drivers affect the fluxes of nutrients and contaminants to the coastal zone, using the driver-pressure-state-impact-response (DPSIR) approach. This is followed by an overview of future research, describing the use of scenarios to simulate future fluxes and provide a consistent framework to evaluate potential policies to improve water quality in the estuary. The Humber catchment is one of eight case studies within a European research project, EUROCAT (EVK1-CT-2000-00044), which aims to achieve integrated catchment and coastal zone management by analysing the response of the coastal sea to changes in fluxes of nutrients and contaminants from the catchments. For the Humber case study, the research focuses on the fluxes of two nutrient elements, N and P, and four metal contaminants, As, Cu, Pb and Zn. The project requires the integration of scientific and socio-economic approaches, bringing together quantitative environmental data garnered for individual river catchments and coastal zones in previous research programmes, and local and regional socio-economic data, to aid decision-makers in their search for integrated and sustainable coastal zone management strategies.


Subharmonics and chaos generation in all-fiber phase modulator: experimental and theoretical analyses with simulation.
We report the results of experimental observation and theoretical analysis of the generation of subharmonic and chaotic signals in a harmonically driven all-fiber phase modulator (PM). When a PM constructed with optical fiber wrapped around a piezoelectric cylinder is driven at high amplitude, we identified that the fiber itself moves with subharmonic frequencies while the piezoelectric cylinder maintains harmonic motion. A theoretical model is presented that is a modification of the model for a bouncing ball on an oscillating table. Some key physical parameters for the model are identified. Potential origins for the discrepancy between the experimental and theoretical analyses are discussed. Ways to suppress the nonlinear effect are also discussed.


Loss of either CD4+ or CD8+ T cells does not affect the magnitude of protective immunity to an intracellular pathogen, Francisella tularensis strain LVS.
Normal mice readily survive a sublethal intradermal (i.d.) infection with Francisella tularensis live vaccine strain (LVS), a model intracellular bacterium, and are strongly protected against subsequent lethal challenge. However, athymic nu/nu mice, which lack mature alphabeta TCR+ T lymphocytes, succumb to i.d. infection within 30 days. Here we characterize the alphabeta T cell subpopulations necessary for both resolution of i.d. infection and generation of optimal protective immunity to LVS. BALB/cByJ mice treated with anti-CD4 or anti-CD8 Abs before i.d. infection survived and cleared bacteria, and anti-CD4- or anti-CD8-treated immune mice survived a very strong i.p. challenge of 10,000 LD50s. Among mutant mice with targeted gene disruptions (knockouts), CD4-, beta2-microglobulin-deficient (which are also CD8-), and gammadelta TCR- mice all resolved a large sublethal i.d. infection. All CD4- and beta2-microglobulin-deficient mice readily survived subsequent lethal i.p. challenge of 10,000 LD50s, even in the absence of specific IgG Abs, as did most (86%) gammadelta TCR- mice. In contrast, alphabeta TCR- mice or alphabeta + gammadelta TCR- mice died about 35 days after i.d. infection. Depletion of gammadelta+ T cells from alphabeta TCR- mice had no effect on mean time to death from i.d. LVS infection. Therefore alphabeta TCR+ cells are required for protection, but either CD4+ or CD8+ T cells are individually sufficient to resolve a large sublethal i.d. LVS infection and to protect against a maximal secondary lethal challenge. These results emphasize the remarkable plasticity of the alphabeta T cell response in protective immunity to intracellular bacteria.


Lipoprotein-Associated Phospholipase A2 Activity Is a Marker of Risk But Not a Useful Target for Treatment in Patients With Stable Coronary Heart Disease.
We evaluated lipoprotein-associated phospholipase A2 (Lp-PLA2) activity in patients with stable coronary heart disease before and during treatment with darapladib, a selective Lp-PLA2 inhibitor, in relation to outcomes and the effects of darapladib in the STABILITY trial. Plasma Lp-PLA2 activity was determined at baseline (n=14 500); at 1 month (n=13 709); serially (n=100) at 3, 6, and 18 months; and at the end of treatment. Adjusted Cox regression models evaluated associations between Lp-PLA2 activity levels and outcomes. At baseline, the median Lp-PLA2 level was 172.4 μmol/min per liter (interquartile range 143.1-204.2 μmol/min per liter). Comparing the highest and lowest Lp-PLA2 quartile groups, the hazard ratios were 1.50 (95% CI 1.23-1.82) for the primary composite end point (cardiovascular death, myocardial infarction, or stroke), 1.95 (95% CI 1.29-2.93) for hospitalization for heart failure, 1.42 (1.07-1.89) for cardiovascular death, and 1.37 (1.03-1.81) for myocardial infarction after adjustment for baseline characteristics, standard laboratory variables, and other prognostic biomarkers. Treatment with darapladib led to a ≈65% persistent reduction in median Lp-PLA2 activity. There were no associations between on-treatment Lp-PLA2 activity or changes of Lp-PLA2 activity and outcomes, and there were no significant interactions between baseline and on-treatment Lp-PLA2 activity or changes in Lp-PLA2 activity levels and the effects of darapladib on outcomes. Although high Lp-PLA2 activity was associated with increased risk of cardiovascular events, pharmacological lowering of Lp-PLA2 activity by ≈65% did not significantly reduce cardiovascular events in patients with stable coronary heart disease, regardless of the baseline level or the magnitude of change of Lp-PLA2 activity. URL: https://www.clinicaltrials.gov. Unique identifier: NCT00799903.


Echocardiography in cardiac arrest: An emergency medicine review.
Cardiac arrest management primarily focuses on optimal chest compressions and early defibrillation for shockable cardiac rhythms. Non-shockable rhythms such as pulseless electrical activity (PEA) and asystole present challenges in management. Point-of-care ultrasound (POCUS) in cardiac arrest is promising. This review provides a focused assessment of POCUS in cardiac arrest, with an overview of transthoracic (TTE) and transesophageal echocardiogram (TEE), uses in arrest, and literature support. Cardiac arrest can be distinguished between shockable and non-shockable rhythms, with management varying based on the rhythm. POCUS provides a diagnostic and prognostic tool in the emergency department (ED), which may improve accuracy in clinical decision-making. Several protocols incorporate POCUS based on different cardiac views. TTE includes parasternal long axis, parasternal short axis, apical 4-chamber, and subxiphoid views, which may be used in cardiac arrest for diagnosis of underlying cause and potential prognostication. TEE is conducted by inserting the probe into the esophagus of intubated patients, with several studies evaluating its use in cardiac arrest. It is associated with few adverse effects, while allowing continued compressions (and evaluation of those compressions) and not interrupting resuscitation efforts. POCUS is a valuable diagnostic and prognostic tool in cardiac arrest, with recent literature supporting its diagnostic ability. TTE can guide resuscitation efforts dependent on the rhythm, though TTE should not interrupt other resuscitation measures. TEE can be useful during arrest, but further studies based in the ED are needed.


Lemon juice has protective activity in a rat urolithiasis model.
The use of herbal medicines (medicinal plants or phytotherapy) has recently gained popularity in Europe and the United States. Nevertheless the exact mechanism of the preventive effects of these products is still far to be clearly established, being its knowledge necessary to successfully apply these therapies to avoid stone formation. The effect of oral lemon juice administration on calcium oxalate urolithiasis was studied in male Wistar rats. Rats were rendered nephrolithic by providing drinking water containing 0.75% ethylene glycol [v/v] (EG) and 2% ammonium chloride [w/v] (AC) for 10 days. In addition to EG/AC treatment, three groups of rats were also gavage-administered solutions containing 100%, 75% or 50% lemon juice [v/v] (6 microl solution/g body weight). Positive control rats were treated with EG/AC but not lemon juice. Negative control rats were provided with normal drinking water, and were administered normal water by gavage. Each group contained 6 rats. After 10 days, serum samples were collected for analysis, the left kidney was removed and assessed for calcium levels using flame spectroscopy, and the right kidney was sectioned for histopathological analysis using light microscopy. Analysis showed that the rats treated with EG/AC alone had higher amounts of calcium in the kidneys compared to negative control rats. This EG/AC-induced increase in kidney calcium levels was inhibited by the administration of lemon juice. Histology showed that rats treated with EG/AC alone had large deposits of calcium oxalate crystals in all parts of the kidney, and that such deposits were not present in rats also treated with either 100% or 75% lemon juice. These data suggest that lemon juice has a protective activity against urolithiasis.


Effect of Ultrasonic Pretreatment on Melon Drying and Computational Fluid Dynamic Modelling of Thermal Profile.
Research background: Drying is one of the most traditional processes of food preservation. Optimizing the process can result in a competitive product on the market regarding its price and quality. A common method in use as a pretreatment to drying is ultrasound. The goal of this work is to analyze different drying methods with and without applying ultrasound (US) pretreatment, on heat and mass transfer, simulating numerically the temperature profile by computational fluid dynamics (CFD). Experimental approach: The melon slices were pretreated with ultrasound for 10 (US10), 20 (US20) and 30 (US30) min at 25 kHz, and the water loss and solid gain were evaluated. Samples were dried at different temperatures (50, 60 and 70 °C). The effective diffusivity was estimated, and experimental data were modelled using empirical models. The airflow in the dryer and the temperature profile in the melon slice were simulated via computational fluid dynamics (CFD). Results and conclusions: Ultrasound pretreatment reduced the drying time from 25% (samples US20 and US30 at 50 °C) to 40% (samples US20 and US30 at 70 °C). The two-term exponential model presented the best fit to the experimental data, and the diffusivity coefficients showed a tendency to increase as the time of exposure of the melon to ultrasonic waves increased. Pretreatment water loss and solid gain behaviour and drying kinetic and diffusion data were used to choose the best experimental conditions to be simulated with CFD. The heat transfer modelling through CFD showed that the temperature distribution along the melon slice was representative. Therefore, the profile obtained via CFD satisfactorily describes the drying process. Novelty and scientific contribution: The use of simulation tools in real processes allows the monitoring and improvement of existing technologies, such as food drying processes, that involve complex mechanisms, making it difficult to obtain some data. Application of CFD in the drying processes of fruits and vegetables is still very recent, being a field little explored. There is no record in the literature that uses CFD in the drying of melon.


Enzyme immunoassay techniques. An overview.
In spite of the great variety of enzyme immunoassays (EIA) they can be classified into two groups 'analyte-observed' and 'reagent-observed' assays, depending on their reaction principle. The latter are favored by use of monoclonal antibodies and are characterized by a greater sensitivity, a larger measuring range, a lower susceptibility to disturbing influences. They can be used only for detection of macromolecules. For heterogeneous EIAs to be used on laboratory scale, simple adsorption of antigens and antibodies is still recommendable though affinity constants decrease by at least one order of magnitude and antibody density at the solid phase and analyte binding capacity are not parallel due to increasing steric hindrance. For this reason, the antibody with the higher affinity constant should therefore always be used as solid-phase antibody. Microparticles used as solid phase for heterogeneous assays, due to their very high binding capacity for the analyte and extremely short diffusion distances, guarantee 'one step' assays of only a few minutes. Of the limited number of enzymes suitable as markers in immunoassays, horseradish peroxidase is the enzyme of choice followed by alkaline phosphatase. Although enzyme and enzyme-labelled reagents are detectable by fluorogenic product measuring with a sensitivity, which is 10-1000 times higher than using chromogenic substrates, the sensitivity of the assays can be increased only by factor 2-10. Labelling enzymes cannot only be covalently bound to the antibody, but also via anti-enzyme antibodies. Pros and cons of the different methods of coupling the enzyme/anti-enzyme complex to analyte-containing immune complexes are discussed. Different EIA variants to detect specific antibodies are reviewed. Among them only capture EIAs permit precise isotype analysis of antibodies of a distinct idiotype. Homogeneous EIAs are widely spread for hapten determination but even variants based on proximal linkage are no alternatives to heterogeneous EIAs for determination of macromolecules. Different parameters are defined which permit to assess the quality of an immunoassay and which should be used in routine assays as internal controls in the laboratory.


Crowding and hydrodynamic interactions likely dominate in vivo macromolecular motion.
To begin to elucidate the principles of intermolecular dynamics in the crowded environment of cells, employing brownian dynamics (BD) simulations, we examined possible mechanism(s) responsible for the great reduction in diffusion constants of macromolecules in vivo from that at infinite dilution. In an Escherichia coli cytoplasm model comprised of 15 different macromolecule types at physiological concentrations, BD simulations of molecular-shaped and equivalent sphere representations were performed with a soft repulsive potential. At cellular concentrations, the calculated diffusion constant of GFP is much larger than experiment, with no significant shape dependence. Next, using the equivalent sphere system, hydrodynamic interactions (HI) were considered. Without adjustable parameters, the in vivo experimental GFP diffusion constant was reproduced. Finally, the effects of nonspecific attractive interactions were examined. The reduction in diffusivity is very sensitive to macromolecular radius with the motion of the largest macromolecules dramatically slowed down; this is not seen if HI dominate. In addition, long-lived clusters involving the largest macromolecules form if attractions dominate, whereas HI give rise to significant, size independent intermolecular dynamic correlations. These qualitative differences provide a testable means of differentiating the importance of HI vs. nonspecific attractive interactions on macromolecular motion in cells.


Inhibition of the type III secretion system of Pseudomonas syringae pv. tomato DC3000 by resveratrol oligomers identified in Vitis vinifera L.
The bacterial type III secretion system (T3SS) is one of the virulence determinants of Gram-negative bacteria through which various effector and virulence proteins are translocated into host cells. We constructed an assay system to screen inhibitors of hrpA gene expression (a structural gene of Hrp pili) in Pseudomonas syringae pv. tomato DC3000. In a plant extract library screening, the root extract of Vitis vinifera L. displayed the most prominent activity. Three resveratrol oligomers, hopeaphenol, isohopeaphenol and ampelopsin A, were identified in grapevine root extract, which significantly reduced the transcription levels of the hrpA, hrpL and hopP1 genes without growth retardation. Additional resveratrol derivatives identified in other plant extracts were also examined for their inhibitory effect on hrpA expression. Another resveratrol oligomer, kobophenol A, also inhibited the transcription of the hrpA gene and other T3SS-related genes, while resveratrol monomers (resveratrol and piceatannol) were not effective. The severity of bacterial specks was reduced by each hopeaphenol, isohopeaphenol and ampelopsin A treatment. These results show the potential of resveratrol derivatives as anti-virulence agents for the control of plant diseases.


A genome-wide association study of saturated, mono- and polyunsaturated red blood cell fatty acids in the Framingham Heart Offspring Study.
Most genome-wide association studies have explored relationships between genetic variants and plasma phospholipid fatty acid proportions, but few have examined apparent genetic influences on the membrane fatty acid profile of red blood cells (RBC). Using RBC fatty acid data from the Framingham Offspring Study, we analyzed over 2.5 million single nucleotide polymorphisms (SNPs) for association with 14 RBC fatty acids identifying 191 different SNPs associated with at least 1 fatty acid. Significant associations (p<1×10(-8)) were located within five distinct 1MB regions. Of particular interest were novel associations between (1) arachidonic acid and PCOLCE2 (regulates apoA-I maturation and modulates apoA-I levels), and (2) oleic and linoleic acid and LPCAT3 (mediates the transfer of fatty acids between glycerolipids). We also replicated previously identified strong associations between SNPs in the FADS (chromosome 11) and ELOVL (chromosome 6) regions. Multiple SNPs explained 8-14% of the variation in 3 high abundance (>11%) fatty acids, but only 1-3% in 4 low abundance (<3%) fatty acids, with the notable exception of dihomo-gamma linolenic acid with 53% of variance explained by SNPs. Further studies are needed to determine the extent to which variations in these genes influence tissue fatty acid content and pathways modulated by fatty acids.


Diabetes Attitudes, Wishes and Needs second study (DAWN2™): cross-national benchmarking of diabetes-related psychosocial outcomes for people with diabetes.
The second Diabetes Attitudes, Wishes and Needs (DAWN2) study aimed to assess psychosocial outcomes in people with diabetes across countries for benchmarking. Surveys included new and adapted questions from validated questionnaires that assess health-related quality of life, self-management, attitudes/beliefs, social support and priorities for improving diabetes care. Questionnaires were conducted online, by telephone or in person. Participants were 8596 adults with diabetes across 17 countries. There were significant between-country differences for all benchmarking indicators; no one country's outcomes were consistently better or worse than others. The proportion with likely depression [WHO-5 Well-Being Index (WHO-5) score ≤ 28] was 13.8% (country range 6.5-24.1%). Diabetes-related distress [Problem Areas in Diabetes Scale 5 (PAID-5) score ≥ 40] was reported by 44.6% of participants (17.2-67.6%). Overall quality of life was rated 'poor' or 'very poor' by 12.2% of participants (7.6-26.1%). Diabetes had a negative impact on all aspects investigated, ranging from 20.5% on relationship with family/friends to 62.2% on physical health. Approximately 40% of participants (18.6-64.9%) reported that their medication interfered with their ability to live a normal life. The availability of person-centred chronic illness care and support for active involvement was rated as low. Following self-care advice for medication and diet was most common, and least common for glucose monitoring and foot examination, with marked country variation. Only 48.8% of respondents had participated in diabetes educational programmes/activities to help manage their diabetes. Cross-national benchmarking using psychometrically validated indicators can help identify areas for improvement and best practices to drive changes that improve outcomes for people with diabetes.


Sexual dysfunction associated with second-generation antidepressants in patients with major depressive disorder: results from a systematic review with network meta-analysis.
Sexual dysfunction (SD) is prevalent in patients with major depressive disorder (MDD) and is also associated with second-generation antidepressants (SGADs) that are commonly used to treat the condition. Evidence indicates under-reporting of SD in efficacy studies. SD associated with antidepressant treatment is a serious side effect that may lead to early termination of treatment and worsening of quality of life. Our objective was to systematically assess the harms of SD associated with SGADs in adult patients with MDD by drug type. We retrieved English-language abstracts from PubMed, EMBASE, the Cochrane Library, PsycINFO, and International Pharmaceutical Abstracts from 1980 to October 2012 as well as from reference lists of pertinent review articles and grey literature searches. Two independent reviewers identified randomized controlled trials (RCTs) of at least 6 weeks' duration and observational studies with at least 1,000 participants. Reviewers abstracted data on study design, conduct, participants, interventions, outcomes and method of SD ascertainment, and rated risk of bias. A senior reviewer checked and confirmed extracted data and risk-of-bias ratings. Random effects network meta-analysis using Bayesian methods for data from head-to-head trials and placebo-controlled comparisons; descriptive analyses calculating weighted mean rates from individual trials and observational studies. Data from 63 studies of low and moderate risk of bias (58 RCTs, five observational studies) with more than 26,000 patients treated with SGADs were included. Based on network meta-analyses of 66 pairwise comparisons from 37 RCTs, most comparisons showed a similar risk of SD among included SGADs. However, credible intervals were wide and included differences that would be considered clinically relevant. We observed three main patterns: bupropion had a statistically significantly lower risk of SD than some other SGADs, and both escitalopram and paroxetine showed a statistically significantly higher risk of SD than some other SGADs. We found reporting of harms related to SD inconsistent and insufficient in some trials. Most trials were conducted in highly selected populations. Search was restricted to English-language only. Because of the indirect nature of the comparisons, the often wide credible intervals, and the high variation in magnitude of outcome, we rated the overall strength of evidence with respect to our findings as low. The current degree of evidence does not allow a precise estimate of comparative risk of SD associated with a specific antidepressant. In the absence of such evidence, clinicians need to be aware of SD as a common adverse event and should discuss patients' preferences before initiating antidepressant therapy.


Viable Compositional Analysis of an Eleven Species Oral Polymicrobial Biofilm.
Polymicrobial biofilms are abundant in clinical disease, particularly within the oral cavity. Creating complex biofilm models that recapitulate the polymicrobiality of oral disease are important in the development of new chemotherapeutic agents. In order to do this accurately we require the ability to undertake compositional analysis, in addition to determine individual cell viability, which is difficult using conventional microbiology. The aim of this study was to develop a defined multispecies denture biofilm model in vitro, and to assess viable compositional analysis following defined oral hygiene regimens. An in vitro multispecies denture biofilm containing various oral commensal and pathogenic bacteria and yeast was created on poly (methyl methacrylate) (PMMA). Denture hygiene regimens tested against the biofilm model included brushing only, denture cleansing only and combinational brushing and denture cleansing. Biofilm composition and viability were assessed by culture (CFU) and molecular (qPCR) methodologies. Scanning electron microscopy and confocal laser scanning microscopy were also employed to visualize changes in denture biofilms following treatment. Combinational treatment of brushing and denture cleansing had the greatest impact on multispecies denture biofilms, reducing the number of live cells by more than 2 logs, and altering the overall composition in favor of streptococci. This was even more evident during the sequential testing, whereby daily sequential treatment reduced the total and live number of bacteria and yeast more than those treated intermittently. Bacteria and yeast remaining following treatment tended to aggregate in the pores of the PMMA, proving more difficult to fully eradicate the biofilm. Overall, we are the first to develop a method to enable viable compositional analysis of an 11 species denture biofilm following chemotherapeutic challenge. We were able to demonstrate viable cell reduction and changes in population dynamics following evaluation of various denture cleansing regimens. Specifically, it was demonstrated that daily combinational treatment of brushing and cleansing proved to be the most advantageous denture hygiene regimen, however, residual organisms still remained within the pores of PMMA surface, which could act as a reservoir for further biofilm regrowth. We have identified an industry need for denture cleansing agents with the capacity to penetrate these pores and disaggregate these complex biofilm consortia.


SFRP2 Is Associated with Increased Adiposity and VEGF Expression.
The aim of this study was to assess depot-specific expression and secretion of secreted frizzled-related protein 2 (sFRP2) by adipose tissue and its effect on adipocyte biology. We measured serum sFRP2 concentrations in 106 patients in vivo to explore its relationship to fat mass, glycaemia and insulin resistance. Expression of sFRP2 in mouse and human tissues was assessed using polymerase chain reaction and Western blot. Western blot confirmed secretion of sFRP2 by adipose tissue into cell culture medium. Effects of recombinant sFRP2 on lipogenesis and preadipocyte proliferation were measured. Preadipocyte expression of the angiogenic genes vascular endothelial growth factor (VEGF) and nuclear factor of activated T-cells 3 (NFATC3) was measured after recombinant sFRP2 exposure. Complementary clinical studies correlating human serum sFRP2 with age, gender, adiposity and insulin secretion were also performed. sFRP2 messenger RNA (mRNA) was expressed in mouse and human adipose tissue. In humans, sFRP2 mRNA expression was 4.2-fold higher in omental than subcutaneous adipose. Omental adipose tissue secreted 63% more sFRP2 protein than subcutaneous. Treatment with recombinant sFRP2 did not impact on lipogenesis or preadipocyte proliferation but was associated with increased VEGF mRNA expression. In human subjects, circulating insulin levels positively correlated with serum sFRP2, and levels were higher in patients with abnormal glucose tolerance (34.2ng/ml) compared to controls (29.5ng/ml). A positive correlation between sFRP2 and BMI was also observed. Circulating sFRP2 is associated with adipose tissue mass and has a potential role to drive adipose angiogenesis through enhanced VEGF expression.


Neto1 and Neto2: auxiliary subunits that determine key properties of native kainate receptors.
Kainate receptors (KARs) are a subfamily of ionotropic glutamate receptors (iGluRs) that mediate excitatory synaptic transmission, regulate neurotransmitter release, and show a remarkably selective distribution in the brain. Compared to other iGluRs, the precise contribution of KARs to brain function is less understood. Unlike recombinant KARs, native KARs exhibit characteristically slow channel kinetics. The underlying explanation for this dissimilar kinetics has remained elusive until recently. New research has identified Neto1 and Neto2 as KAR auxiliary subunits that determine unique properties of synaptic KARs, including their slow kinetics and high affinity for agonist. Whether these auxiliary subunits regulate KAR trafficking and targeting at the synapse is less clear. By regulating channel gating, Neto1 and Neto2 can increase the diversity of KAR functional properties. These auxiliary subunits may represent a starting point for a better understanding of the role played by neuronal KARs under normal and pathological conditions, but also, they may provide an alternative target for the development of new drugs regulating KARs and brain function.


Histopathology and functional correlations in a patient with a mutation in RPE65, the gene for retinol isomerase.
Here the authors describe the structural features of the retina and retinal pigment epithelium (RPE) in postmortem donor eyes of a 56-year-old patient with a homozygous missense RPE65 mutation (Ala132Thr) and correlate the pathology with the patient's visual function last measured at age 51. Eyes were enucleated within 13.5 hours after death. Representative areas from the macula and periphery were processed for light and electron microscopy. Immunofluorescence was used to localize the distribution of RPE65, rhodopsin, and cone arrestin. The autofluorescence in the RPE was compared with that of two normal eyes from age-similar donors. Histologic examination revealed the loss of rods and cones across most areas of the retina, attenuated retinal vessels, and RPE thinning in both eyes. A small number of highly disorganized cones were present in the macula that showed simultaneous labeling with cone arrestin and red/green or blue opsin. RPE65 immunoreactivity and RPE autofluorescence were reduced compared with control eyes in all areas studied. Rhodopsin labeling was observed in rods in the far periphery. The optic nerve showed a reduced number of axons. The clinical findings of reduced visual acuity, constricted fields, and reduced electroretinograms (ERGs) 5 years before death correlated with the small number of cones present in the macula and the extensive loss of photoreceptors in the periphery. The absence of autofluorescence in the RPE suggests that photoreceptor cells were probably missing across the retina for extended periods of time. Possible mechanisms that could lead to photoreceptor cell death are discussed.


Comparative Analysis of Different Methods of Modeling the Thermal Effect of Circulating Blood Flow During RF Cardiac Ablation.
Our aim was to compare the different methods of modeling the effect of circulating blood flow on the thermal lesion dimensions created by radio frequency (RF) cardiac ablation and on the maximum blood temperature. Computational models were built to study the temperature distributions and lesion dimensions created by a nonirrigated electrode by two RF energy delivery protocols (constant voltage and constant temperature) under high and low blood flow conditions. Four methods of modeling the effect of circulating blood flow on lesion dimensions and temperature distribution were compared. Three of them considered convective coefficients at the electrode-blood and tissue-blood interfaces to model blood flow: 1) without including blood as a part of the domain; 2) constant electrical conductivity of blood; and 3) temperature-dependent electrical conductivity of blood (+2%/°C). Method 4) included blood motion and was considered to be a reference method for comparison purposes. Only Method 4 provided a realistic blood temperature distribution. The other three methods predicted lesion depth values similar to those of the reference method (differences smaller than 1 mm), regardless of ablation mode and blood flow conditions. Considering the aspects of lesion size and maximum temperature reached in blood and tissue, Method 2 seems to be the most suitable alternative to Method 4 in order to reduce the computational complexity. Our findings could have an important implication in future studies of RF cardiac ablation, in particular, in choosing the most suitable method to model the thermal effect of circulating blood.


Asymmetric synthesis of new γ-butenolides via organocatalyzed epoxidation of chalcones.
γ-Butenolides have been recognized as an important structural framework in a number of natural products and medicinally important agents. In this work we describe a new metal-free sequential strategy for the asymmetric synthesis of substituted γ-butenolides having epoxychalcones as the advanced intermediate. Using the optimized reaction conditions, we were able to carry out the three-step sequence, epoxidation, olefination and hydrolysis, with only one single chromatographic purification of the final product, furnishing new enantiomerically enriched γ-butenolides in moderate overall yield and good enantiomeric excess.


High Frequency Components of Hemodynamic Shear Stress Profiles are a Major Determinant of Shear-Mediated Platelet Activation in Therapeutic Blood Recirculating Devices.
We systematically analyzed the relative contributions of frequency component elements of hemodynamic shear stress waveforms encountered in cardiovascular blood recirculating devices as to overall platelet activation over time. We demonstrated that high frequency oscillations are the major determinants for priming, triggering and yielding activated "prothrombotic behavior" for stimulated platelets, even if the imparted shear stress has low magnitude and brief exposure time. Conversely, the low frequency components of the stress signal, with limited oscillations over time, did not induce significant activation, despite being of high magnitude and/or exposure time. In vitro data were compared with numerical predictions computed according to a recently proposed numerical model of shear-mediated platelet activation. The numerical model effectively resolved the correlation between platelet activation and the various frequency components examined. However, numerical predictions exhibited a different activation trend compared to experimental results for different time points of a stress activation sequence. With this study we provide a more fundamental understanding for the mechanobiological responsiveness of circulating platelets to the hemodynamic environment of cardiovascular devices, and the importance of these environments in mediating life-threatening thromboembolic complications associated with shear-mediated platelet activation. Experimental data will guide further optimization of the thromboresistance of cardiovascular implantable therapeutic devices.


Prevalence of serological markers and nucleic acid for blood-borne viral infections in blood donors in Al-Baha, Saudi Arabia.
Data on blood-borne viral infections in some regions in Saudi Arabia remain scarce. This study investigates the prevalence of serological markers and nucleic acid for blood-borne viruses among blood donors in Al-Baha, Kingdom of Saudi Arabia. In this cross-sectional study, 2,807 donors who donated blood between January 2009 and November 2011 were investigated for blood-borne viral serological markers including HBsAg, anti-HBc, anti-HBs, anti-HCV, anti-HIV, and anti-HTLVI/III in addition to viral nucleic acid. All donors were males between 16 to 66 years of age (mean: 31.5 ± 9.3 years). Viral nucleic acid and/or serological markers were detected in a total of 36 (1.3%) donors; of them, 26 (72.2%) had nucleic acid concomitant with serological markers, 6 (16.7%) had only viral nucleic acid, while 4 (11.1%) had only serological markers. Of all donors, 22 (0.8%) had HBsAg, 227 (8.0%) had anti-HBc, 157 (5.0%) had anti-HBs, 2,577 (91.8%) had no HBV markers, 2 (0.07%) had anti-HIV, 1 (0.04%) had anti-HCV, and 1 (0.04%) had anti-HTLVI/II. The donors who were born during HBV vaccination era showed no HBsAg (0.0%; p = 0.052), lower rates of anti-HBc (1.5%; p < 0.001) and anti-HBs (0.7%; p < 0.001), while the majority had no HBV markers (98.5%; p < 0.001). Combined viral nucleic acid and serological testing of donated blood enhances blood safety. The absence of HBV markers among donors suggests susceptibility or declined anti-HBs levels. Thus, HBV revaccination or a vaccine boost among adolescents and adults might be indispensable.


Antiplatelet therapy in the primary prevention of cardiovascular disease in patients with chronic obstructive pulmonary disease: a randomised controlled proof-of-concept trial.
The APPLE COPD-ICON2 trial is a prospective 2×2 factorial, double-blinded proof-of-concept randomised controlled trial targeting patients with chronic obstructive pulmonary disease (COPD) without prior history of cardiovascular disease. The primary goal of this trial is to investigate if treatment with antiplatelet therapy will produce the predefined cut-off of platelet inhibition measured using the Multiplate test in COPD patients. Eligible patients were randomised to aspirin plus placebo, ticagrelor plus placebo, aspirin plus ticagrelor or placebo only for 6 months. The primary outcome comprises inhibition (binary response) of arachidonic acid- (ASPI test, cut-off <40) and adenosine diphosphate- (ADP test, cut-off <46) induced platelet aggregation at 6 months. 543 patients were screened and 120 patients were recruited with mean age of 67.5 years; 47.5% patients were male. The per-protocol ASPI test response rate to aspirin was 68.3% (95% CI 52.3-80.9%). The per-protocol ADP test response rate to ticagrelaor was 68.8% (95% CI 50.4-82.6%). Platelet response to antiplatelet therapy with aspirin and ticagrelor was not observed in nearly one-third of COPD patients without prior history of cardiovascular disease. These findings support the high pro-thrombotic milieu and the need for further research to determine the effect of antiplatelet/antithrombotic therapy on cardiovascular morbidity and mortality in COPD patients.


Origin of the nonadhesive properties of fibrinogen matrices probed by force spectroscopy.
The deposition of a multilayered fibrinogen matrix on various surfaces results in a dramatic reduction of integrin-mediated cell adhesion and outside-in signaling in platelets and leukocytes. The conversion of a highly adhesive, low-density fibrinogen substrate to the nonadhesive high-density fibrinogen matrix occurs within a very narrow range of fibrinogen coating concentrations. The molecular events responsible for this transition are not well understood. Herein, single-cell and molecular force spectroscopy were used to determine the early steps in the formation of nonadhesive fibrinogen substrates. We show that the adsorption of fibrinogen in the form of a molecular bilayer coincides with a several-fold reduction in the adhesion forces generated between the AFM tip and the substrate as well as between a cell and the substrate. The subsequent deposition of new layers at higher coating concentrations of fibrinogen results in a small additional decrease in adhesion forces. The poorly adhesive fibrinogen bilayer is more extensible under an applied tensile force than is the surface-bound fibrinogen monolayer. Following chemical cross-linking, the stabilized bilayer displays the mechanical and adhesive properties characteristic of a more adhesive fibrinogen monolayer. We propose that a greater compliance of the bi- and multilayer fibrinogen matrices has its origin in the interaction between the molecules forming the adjacent layers. Understanding the mechanical properties of nonadhesive fibrinogen matrices should be of importance in the therapeutic control of pathological thrombosis and in biomaterials science.


Towards a phylogeny and definition of species at the molecular level within the genus Mycobacterium.
16S rRNA sequences from Mycobacterium tuberculosis, M. avium, M. gastri, M. kansasii, M. marinum, M. chelonae, M. smegmatis, M. terrae, M. gordonae, M. scrofulaceum, M. szulgai, M. intracellulare, M. nonchromogenicum, M. xenopi, M. malmoense, M. simiae, M. flavescens, M. fortuitum, and M. paratuberculosis were determined and compared. The sequence data were used to infer a phylogenetic tree, which provided the basis for a systematic phylogenetic analysis of the genus Mycobacterium. The groups of slow- and fast-growing mycobacteria could be differentiated as distinct entities. We found that M. simiae occupies phylogenetically an intermediate position between these two groups. The phylogenetic relatedness within the slow-growing species did not reflect the Runyon classification of photochromogenic, scotchromogenic, and nonchromogenic mycobacteria. In general, the phylogenetic units identified by using rRNA sequences confirmed the validity of phenotypically defined species; an exception was M. gastri, which was indistinguishable from M. kansasii when this kind of analysis was used.


Dye spread through gap junctions in the corneal epithelium of the rabbit.
Microelectrode dye injection of 5,6-carboxyfluorescein was used to investigate gap junctional communication in the corneal epithelium. Dye injection started in the superficial layer and proceeded stepwise into the underlying epithelial layers until spread was observed. Intracellular [Ca2+] was manipulated by exposing the cornea to the calcium ionophore A23187 (global increase) or by increasing the [Ca2+] in the injection electrode (source cell increase). Intracellular pH was manipulated by exposing the cornea to nigericin in a low-pH KCI Ringer's (global decrease) or by lowering the pH in the injection electrode (source cell decrease). Heptanol was tested for its ability to uncouple gap junctions. Gap junctional communication was based on the layer at which spread was first observed and on the apparent dye travel distance from the point of injection. Control dye spread occurred, on average, in the third layer from the surface. Increased [Ca2+] in the source cell resulted in an initial spread occurring in the second layer. Globally increasing [Ca2+] with A23187 resulted in no change in the average initial spread layer. Lowering intracellular pH of the source cell did not affect the initial dye spread layer. Globally lowering intracellular pH resulted in significant gap junctional inhibition in a time-dependent manner. Dye spread distance was not significantly affected by [Ca2+] or pH manipulations. Heptanol (2.5 mM) completely inhibited dye coupling. All cell layers of the corneal epithelium contain functional gap junctions, although it appears that intercellular communication in the superficial layers does not occur under our control conditions. Intercellular communication through these junctions can be altered by various manipulations of [Ca2+] and pH.


Methodology for tissue sample collection within a translational sub-study of the CHHiP trial (CRUK/06/016), a large randomised phase III trial in localised prostate cancer.
This article presents the methodology for tissue sample collection in Trans-CHHiP, the main translational study within the CHHiP (Conventional or Hypofractionated High dose intensity modulated radiotherapy in Prostate cancer, ISRCTN 97182923) trial. The CHHiP trial randomised 3216 men with localised prostate cancer to 3 different radiotherapy fractionation schedules. Trans-CHHiP aims to identify biomarkers of fraction sensitivity. We outline the process of tissue collection, including central review by a study-specific specialist uropathologist and comparison of the centrally-assigned Gleason grade group with that assigned by the recruiting-centre pathologist. 2047 patients provided tissue from 107 pathology departments between August 2012 and April 2014. A highly motivated Clinical Trials Unit chasing samples and a central Trans-CHHiP group that regularly reviewed progress were important for successful sample collection. Agreement in Gleason grade group assigned by the recruiting centre pathologist and the central study-specific uropathologist occurred in 886 out of 1854 (47.8%) cases. Key lessons learned were the need for prospective consent for tissue collection when recruiting patients to the main trial, and the importance of Material Transfer Agreement (MTA) integration into the initial trial site agreement. This methodology enabled collection of 2047 patient samples from a large randomised radiotherapy trial. Central pathological review is important to minimise subjectivity in Gleason grade grouping and the impact of grade shift.


P2X7 blockade attenuates mouse liver fibrosis.
P2X7 is important in inflammation and tissue injury. The aim of the present study was to investigate the effect of P2X7 inhibition, using a specific inhibitor (A438079) to prevent the development of liver injury and fibrosis in a mouse model of liver fibrosis. The mouse liver fibrosis model was induced by carbon tetrachloride (CCl4). Mice received subcutaneous administration of vehicle (saline/olive oil), CCl4 or subcutaneous CCl4 and A438079. The pro‑inflammatory and pro‑fibrotic factors were determined by western blot analysis. The biochemistry, histopathology, collagen deposition and nuclear factor‑κB (NF‑κB) activity were also analyzed. Chronic CCl4 treatment resulted in liver injury and collagen accumulation. The expression levels of P2X7, pro‑inflammatory and pro‑fibrotic mediators, and the activity of NF‑κB were markedly increased. Treatment with A438079 significantly inhibited CCl4‑induced P2X7 expression, and attenuated CCl4‑induced liver injury and the inflammatory response. P2X7 blockade also significantly reduced the formation of collagen in the liver and the expression of α-smooth muscle actin and transforming growth factor‑β1. This study demonstrated that P2X7 inhibition attenuated liver injury and fibrosis in a mouse model. Thus, P2X7 is a potential novel therapeutic target for liver injury and fibrosis.


A Novel Nonsense Mutation in PANK2 Gene in Two Patients with Pantothenate Kinase-Associated Neurodegeneration.
Pantothenate kinase- associated neurodegeneration (PKAN) syndrome is a rare autosomal recessive disorder characterized by progressive extrapyramidal dysfunction and iron accumulation in the brain and axonal spheroids in the central nervous system. It has been shown that the disorder is caused by mutations in PANK2 gene which codes for a mitochondrial enzyme participating in coenzyme A biosynthesis. Here we report two cases of classic PKAN syndrome with early onset of neurodegenerative disorder. Mutational analysis has revealed that both are homozygous for a novel nonsense mutation in PANK2 gene (c.T936A (p.C312X)). The high prevalence of consanguineous marriages in Iran raises the likelihood of occurrence of autosomal recessive disorders such as PKAN and necessitates proper premarital genetic counseling. Further research is needed to provide the data on the prevalence of PKAN and identification of common PANK2 mutations in Iranian population.


Peptide-Based Identification of Phytophthora Isolates and Phytophthora Detection in Planta.
Phytophthora is arguably one of the most damaging genera of plant pathogens. This pathogen is well suited to transmission via the international plant trade, and globalization has been promoting its spread since the 19th century. Early detection is essential for reducing its economic and ecological impact. Here, a shotgun proteomics approach was utilized for Phytophthora analysis. The collection of 37 Phytophthora isolates representing 12 different species was screened for species-specific peptide patterns. Next, Phytophthora proteins were detected in planta, employing model plants Solanum tuberosum and Hordeum vulgare. Although the evolutionarily conserved sequences represented more than 10% of the host proteome and limited the pathogen detection, the comparison between qPCR and protein data highlighted more than 300 protein markers, which correlated positively with the amount of P. infestans DNA. Finally, the analysis of P. palmivora response in barley revealed significant alterations in plant metabolism. These changes included enzymes of cell wall metabolism, ROS production, and proteins involved in trafficking. The observed root-specific attenuation in stress-response mechanisms, including the biosynthesis of jasmonates, ethylene and polyamines, and an accumulation of serotonin, provided the first insight into molecular mechanisms behind this particular biotic interaction.


Re-evaluation of hepatitis B virus clinical phases by systems biology identifies unappreciated roles for the innate immune response and B cells.
To identify immunological mechanisms that govern distinct clinical phases of a chronic hepatitis B virus (HBV) infection-immune tolerant (IT), immune active (IA), inactive carrier (IC), and hepatitis B e antigen (HBeAg)-negative (ENEG) hepatitis phases-we performed a systems biology study. Serum samples from untreated chronic HBV patients (n = 71) were used for multiplex cytokine measurements, quantitative hepatitis B surface antigen (HBsAg), HBeAg levels, HBV genotype, and mutant analysis. Leukocytes were phenotyped using multicolor flow cytometry, and whole-blood transcriptome profiles were generated. The latter were compared with liver biopsy transcriptomes from IA (n = 16) and IT (n = 3) patients. HBV viral load as well as HBeAg and HBsAg levels (P < 0.001), but not leukocyte composition, differed significantly between distinct phases. Serum macrophage chemotactic protein 1, interleukin-12p40, interferon (IFN)-gamma-inducible protein 10, and macrophage inflammatory protein 1 beta levels were different between two or more clinical phases (P < 0.05). Comparison of blood transcriptomes identified 64 differentially expressed genes. The gene signature distinguishing IA from IT and IC patients was predominantly composed of highly up-regulated immunoglobulin-encoding genes. Modular repertoire analysis using gene sets clustered according to similar expression patterns corroborated the abundant expression of B-cell function-related genes in IA patients and pointed toward increased (ISG) transcript levels in IT patients, compared to subsequent phases. Natural killer cell activities were clustered in clinical phases with biochemical liver damage (IA and ENEG phases), whereas T-cell activities were higher in all phases, compared to IT patients. B-cell-related transcripts proved to be higher in biopsies from IA versus IT patients. HBV clinical phases are characterized by distinct blood gene signatures. Innate IFN and B-cell responses are highly active during the IT and IA phases, respectively. This suggests that the presumed immune tolerance in chronic HBV infections needs to be redefined.